The invention relates generally to the fields of biosensors and drug screening.
Many proteins exhibit altered properties upon binding of a specific ligand with a specialized domain of the protein. Such specialized domains are generally referred to as “ligand binding domains” (LBDs). The details of the mechanism by which ligand binding alters the properties of a protein are not well understood for all ligand binding-modulated proteins. Nonetheless, it is known that, for many such proteins, binding of a ligand with a LBD alters the three-dimensional conformation of the protein such that the conformation of another domain (i.e., one distinct from the LBD) of the protein is altered in a way that affects the catalytic or binding characteristics of the domain. By way of example, nuclear hormone receptors such as the estrogen receptor are activated upon ligand binding such that the activated receptor can modulate gene expression.
The nuclear hormone receptors are a family of ligand-activated eukaryotic transcription factors that control the expression of genes in response to the presence of small-molecule hormones or hormone-like compounds (Aranda et al., 2001, Physiol. Rev. 81:1269-1304). The estrogen, androgen, thyroid hormone, progesterone and vitamin D receptors are some well known members of this receptor superfamily. They comprise a very important class of drug targets, as their function has been linked to a broad spectrum of diseases, including breast, endometrial and prostate cancer, leukemia, cardiovascular diseases, osteoporosis and inflammations (Bourguet et al., 2000, Trends Pharmacol. Sci. 21:381-388; Riggs et al., 2003, N. Engl. J. Med. 348:618-629). Approximately 2% of the drug targets of current therapies belong to this receptor superfamily (Drews, 2000, Science 287:1960-1964), while 10% of the prescribed drugs are intended to modulate the function of those transcription factors. Discovery of novel compounds with the ability to modulate these targets is an important goal for the pharmaceutical industry.
Currently used screening strategies for identifying novel compounds with hormone-mimicking properties include in vitro competitive binding assays with radiolabeled hormones, transcriptional activation assays with various reporter proteins in Saccharomyces cerevisiae, and animal cell proliferation assays (Joyeux et al., 1997, Anal. Biochem. 249:119-130; Zacharewski, 1997, Environ. Sci. Technol. 31:613-623). One such example is the E-screen, where monitoring of the proliferation of human breast cancer cells in the presence of test compounds is used to evaluate their estrogenicity (Soto et al., 1995, Environ. Health Perspect. 103(Suppl 7):113-122). These screening approaches are generally complex, time-consuming, and expensive. There is a need for simpler, cheaper, and more rapidly performable assays for lead identification and characterization.
Naturally-occurring proteins often exhibit high binding specificity. Proteins which bind specifically with a particular ligand can therefore be used as a sensor or detector for the ligand if ligand binding can be observed. Furthermore, in the field of pharmacology, it is desirable to identify compounds that can interact with the LBD of a protein involved in a biological process, all the more so if the consequence of binding of the compound with the LBD can be detected. If the compound identification process can be performed rapidly and inexpensively, then collections of many compounds can be screened to identify individual compounds and types of compounds effective for modulating the biological process. Furthermore, if the detection method can be linked to a screenable or selectable phenotype in growing cells, then it can facilitate evolution of new drug compounds that have not been detected in any natural product.
The present invention satisfies these and other needs in the art by providing a method of assessing ligand binding that is relatively simple, cheap, and physiologically relevant.